Abstract

The camptothecins are a group of anticancer agents with a unique
mechanism of action: poisoning of eukaryotic DNA topoisomerase I.
9-aminocamptothecin (9-AC), a potent water-insoluble derivative of
camptothecin, is currently undergoing clinical testing. The kinetics of
the active derivative 9-AC lactone in cell culture media was defined,
and then 9-AC cytotoxicity against human breast (MCF-7), bladder
(MGH-U1), and colon (HT-29) cancer cell lines was studied. The
relationship between cytotoxic effects, drug concentration, and
exposure time was then explored. For all of the three cell lines, 9-AC
cytotoxicity increased with both higher drug concentrations and longer
exposure times. However, when the duration of exposure was less than
24 h, cytotoxicity was limited and less than 1 log of cell killing
occurred, even with very high drug concentrations. Minimal cell killing
was also observed unless 9-AC concentrations exceeded a threshold of
2.7 nm. No fixed relationship between the survival fraction
and the area under the drug concentration-time curve could be modeled
that would fit all of the three cell lines. However, data for the three
cell lines from the multiple exposure time experiments were fitted very
well to the pharmacodynamic model
Cnt =
k (r2,0.90–0.99),
where C is the drug concentration, n is
the drug concentration coefficient, and t is the
exposure time. For the three cell lines, to kill 1 log of cells,
0.30 < n < 0.85, which indicated that
duration of exposure was more important than concentration. Our data
support the use of 9-AC by infusion for 24 h or longer in clinical
studies providing target plasma concentrations can be achieved.

INTRODUCTION

9-AC2
is a water-insoluble derivative of CPT, an anticancer agent derived
from the stemwood of the Chinese tree Camptotheca acuminata
by Monroe Wall and colleagues in 1966 (1)
. CPT was found
to inhibit the growth of a wide range of experimental tumors in
vitro(2, 3, 4, 5)
. Because CPT was water-insoluble, the
water-soluble derivative sodium CPT was used in early clinical trials.
Clinical evaluation was discontinued in the early 1970s because of
unpredictable serious toxicity and low antitumor activity (6, 7)
. Subsequent studies found that an intact lactone ring (E
ring) with an “S” stereoisomeric configuration at carbon 20 is
essential for antitumor activity (4, 8, 9)
. The CPT
lactone undergoes hydrolysis to produce a water-soluble hydroxy acid or
carboxylate form, which is much less potent as an inhibitor of
topoisomerase I and as an anticancer agent (4, 8, 9, 10)
. The lactone and carboxylate forms of CPTs exist in a
pH-dependent equilibrium. Interest in the CPTs was renewed in the late
1980s after reports that the enzyme topoisomerase I was its major
cellular target (11)
. A series of CPT derivatives with
increased potency and lower toxicities compared with the parent
compound have been synthesized and evaluated (4, 12, 13)
.
Derivatives of CPT currently in clinical study include 9-AC, topotecan,
9-nitrocamptothecin, and irinotecan (CPT-11).

There has been considerable research effort into the interdependent
relationship of both drug concentration and time on antitumor activity.
Of particular interest is whether clinical response can be correlated
with a function of concentration and time such as the plasma AUC
(14)
. Several studies have found that the correlation
between cytotoxicity and C × t is often imperfect
(15, 16)
. One reason for failure to observe a consistent
dependence of antitumor activity on C × t
is explained by the equation Cn ×
t = k(17)
. In this equation,
C is the drug concentration, t is the exposure
time, n is the drug concentration coefficient, and
k is the drug exposure constant. When the exponent
n is <1, this indicates a greater importance for exposure
time, whereas n > 1 indicates a greater importance for
concentration (18, 19)
. This relationship was first
proposed to describe the effect of disinfectants on bacteria. The
relevance of this relationship to antitumor agents was realized by
Skipper (17)
, who used this relationship to describe the
effect of antitumor agents on reducing a leukemic cell population.
Recently, Adam et al.(18)
proposed the use of
this equation to calculate the Cmin
and tmin required to achieve a
therapeutic end point. These parameters represent the minimum
conditions required to produce a certain level of antitumor effect and
are useful when comparing the effect of different antitumor agents or a
single agent on different patients considering both the extracellular
concentration and exposure time rather than concentration alone.

Although a number of studies have examined the cytotoxicity of 9-AC
against human cancer cells (20, 21)
, few studies have
studied the relationship between cytotoxicity and drug concentrations,
especially the relationship between the cytotoxicity, concentration,
and exposure time. Researchers have found that exposure time is
important for the cytotoxicity of 9-AC; however, the exact model of the
Effect-Concentration-Time relationship has not yet been established.
The main purpose of this project was to investigate the PK/PD
relationship of 9-AC against human cancer cells. Once this relationship
is established, it would provide a useful PD framework for additional
in vitro and in vivo studies.

MATERIALS AND METHODS

Cells.

Experiments were performed with the human cancer cell lines MCF-7
(breast), MGH-U1, (bladder), and HT-29 (colon). All of the lines were
obtained originally from the American Type Culture Collection
(Rockville, MD). Cells were maintained routinely at 37°C in a
humidified 5% CO2 incubator as monolayer
cultures in α-MEM with ribonucleosides and deoxyribonucleosides
growth media (provided by Media Department, Ontario Cancer
Institute, Ontario, Canada) supplemented with 10% fetal bovine serum
and the antibiotics penicillin and streptomycin.

Reagents and Chemicals.

9-AC was provided by Pharmacia Inc. (Albuquerque, NM). CPT was a gift
of Dr. M. C. Wani (Research Triangle Institute, Research Triangle
Park, NC). For all of the experiments, CPT and 9-AC were dissolved in
dehydrated DMSO and stored at −70°C until used.
high-performance liquid chromatography-grade methanol was purchase from
VWR Scientific (Toronto, Ontario, Canada). All of the other chemicals
were molecular biology grade and were purchased from Sigma (St. Louis,
MO) unless otherwise specified.

The Kinetics of 9-AC in Cell Culture Media.

Kinetic studies were performed in cell-free culture media at 37°C,
5% CO2, at drug concentrations of 13.7
nm (5 ng/ml) and 137 nm (50 ng/ml). The media
was left in the incubator overnight to allow the media to have the same
condition as in cell cytotoxicity studies. Stock solutions (30 μl) of
9-AC at 13.7 μg/ml or 137 μg/ml were added to 30 ml of medium. A
1-ml sample was taken at 5, 10, 15, 20, 30, 45, and 60 min and at 2, 4,
8, 12, 24, 48, 72, and 240 h after the drug was added. A
high-performance liquid chromatography method developed by Takimoto
et al., (22)
was used to extract and quantitate
the 9-AC lactone, using CPT as the internal standard. The total 9-AC
(lactone + carboxylate) concentration was extracted by a slight
modification of the lactone extraction procedure. Media aliquots
(0.1-ml) were acidified with 0.9 ml of 8.5% phosphoric acid and
incubated at room temperature to allow for complete conversion of 9-AC
carboxylate to 9-AC lactone. Then the samples were analyzed by the same
method used for 9-AC lactone. The 9-AC peak was detected by using a
Shimadzu RF-10A fluorescent detector at an excitation wavelength of 365
nm and an emission wavelength of 440 nm.

Cytotoxicity of 9-AC.

The cytotoxicity of 9-AC was assessed by clonogenic assay (23, 24)
. Exponentially growing cells were resuspended in media, cell
number was determined using an electronic counter, and 100–250 cells
were inoculated in triplicate onto 60 15-mm dishes containing 5 ml of
medium. After an overnight incubation, 5 μl of 9-AC stock solutions
were added to the dishes to achieve final concentrations of 0, 0.27,
1.37, 2.74, 13.7, 27.4, 137, and 274 nm. After 4-, 8-, 12-,
24-, 48-, 72-, and 240-h exposures, medium was removed by aspiration
and fresh medium was added to the dishes. Percentage of survival at
each drug concentration with different exposure time was determined
from the ratio of the number of the colonies in the drug-treated
sample:the number in the control (DMSO vehicle-treated) sample.

Kinetic Analysis of the Hydrolysis of 9-AC Lactone.

The concentration of intact lactone (C) versus
time (t) date for the 9-AC were fitted to the equation below
by the method of nonlinear least squares (25)
:
where the parameter k1 is the
pseudo first-order rate constant of hydrolysis of the lactone ring,
from which t1/2 of hydrolysis can be
calculated by dividing 0.693 by the rate constant,
k1. The parameter “a”
corresponds to the concentration of the intact lactone form at
equilibrium, and (a + b) equals the total intact
lactone form of drug at t = 0 (when 9-AC was first
added to media). Fitting of experimental data to the equation was
accomplished using Scientist software (MicroMath Scientific Software,
Salt Lake City, UT).

The observed equilibrium constant
(kobs), for the conversion of the
lactone to its corresponding ring opened species is calculated from
(opened)eq/(closed)eq,
whereas the percentage opened was calculated from the fraction
(opened)eq/(total)eq(26)
. The value of kobs
was obtained from the average of three independent experiments.

The Effect-Concentration (E-C)
Relationship.

Fitting the experimental data to each of the following equations, we
analyzed the relationship between cytotoxicity (E) and drug
concentration (C).
where E is the survival percentage; C is the
9-AC concentration; Emax is the
maximum drug effect; EC50 is the
concentration that produces one-half of the maximum effect; and
n is the Hill constant, which describes the shape of the
curve.

In log-linear E-C model, a is a slope
parameter and b is a constant. In the case in which the
maximum effect was not achieved, the data were fitted to the log-linear
model. If maximum effect was achieved, the data were fitted to
Sigmoidal Emax model using nonlinear
least squares regression Scientist software and values for
IC50, IC90, n,
and Emax were determined.

The Effect-Concentration-Time
(E-C-t) Relationship.

The 9-AC AUC for each different exposure time was calculated
using the LAGRAN V1.0B software (University of Alberta, Edmonton,
Alberta, Canada). Then the relationship between the AUC of 9-AC
lactone and the cytotoxicity of 9-AC was analyzed.

The relationship between drug concentration, exposure time, and
cytotoxicity was fit to the following PD model (18)
:
In this model, C, t, n, and
k are the drug concentration, exposure time, drug
concentration coefficient, and the drug exposure constant,
respectively. n and k were determined from a plot
of the isotoxic concentrations IC50 or
IC90versus exposure time using the
least squares nonlinear regression using the Scientist software package
(MicroMath Scientific Software). n <1 indicates that the
duration of exposure is more important than the concentration. The
minimum concentration (Cmin) and time
(tmin) to reach certain cytotoxicity
were also calculated using the following equations:

Cell Cycle Analysis.

Flow cytometry with propidium iodide staining was performed prior to
and after 4- and 24-h drug exposures at drug concentrations of 13.7
nm (5 ng/ml) and 137 nm (50 ng/ml) by the
procedure of Kastan et al.(27)
. Briefly, cells were
harvested and fixed on ice in 80% ethanol for 1 h. Prior to flow
cytometry, cells were washed with d-PBS
(Ca2+-Mg2+-free-PBS)
treated with 1 mg/ml RNase and stained with 50 μg/ml propidium iodide
for at least 60 min. Samples were analyzed on a FACScan flow cytometer
(Becton Dickinson, Mansfield, MS). Data were gated on light scatter
before the recording of a histogram composed of 10,000 cells. The
percentage of cells in each phase of the cell cycle was quantified
using the MODfit software package (Verity Software House Inc., Topsham,
ME). Each value shown in the results represents the mean ±
SD of three or more independent experiments.

RESULTS

The Kinetics of 9-AC.

Fig. 1⇓
depicts changes in lactone concentration as a function of time for 137
nm 9-AC in media at 37°C and 5%
CO2. The lactone concentration decreased rapidly
in the first 2 h and reached equilibrium in media after a 6-h
incubation. This equilibrium remained constant for the 10 days that
this was studied. The total concentration of 9-AC (lactone +
carboxylate) did not change significantly during the 240-h incubation
(Fig. 1)⇓
.

Stability of 9-AC lactone and total forms in
cell culture media. 9-AC lactone was added to media at a concentration
of 137 nm and incubated at 37°C with 5% CO2.
Each point represents the average of at least three independent kinetic
runs with the same sampling schedules. (•) observed concentration of
9-AC lactone; (—) fitted kinetic profile of 9-AC lactone; (▪)
observed concentration of 9-AC total form; (… ) initial
concentration of 9-AC total form.

The concentration of 9-AC lactone (C) versus time
(t) data were fitted to =
a + b
Exp(−k1t) by the method of
nonlinear least squares (Fig. 1)⇓
. The pseudo first-order rate constant
of hydrolysis of 137 nm 9-AC lactone was
calculated to be 0.724 h1. The result showed that
the hydrolysis of 9-AC lactone proceeded with a
t1/2 of approximately 57 min and achieved a
final lactone:carboxylate equilibrium ratio of 35:65. The observed
equilibrium constant, kobs, for the
conversion of the lactone to its corresponding ring opened species was
calculated to be 1.857.

The kinetic profile of 13.7 nm 9-AC lactone in cell culture
media was similar to that of 137 nm 9-AC, which indicated
that the rate of this process was independent of drug concentration
over the ranges examined in this study. Its pseudo first-order rate
constant was 0.968 h−1, and the final
lactone:carboxylate equilibrium ratio was 37:63. The observed
equilibrium constant, kobs, was
calculated to be 1.703.

Cytotoxicity of 9-AC.

The survival percentages for the MCF-7, MGH-U1, and HT-29 cell lines at
each 9-AC concentration with different exposure times all demonstrated
minimal cytotoxicity until concentrations in excess of 3–5
nm were used. The data for cell line HT-29 is shown in Fig. 2⇓
. All of the three cell lines displayed some sensitivity to 9-AC (Table 1)⇓
. When a maximum effect (100%) was approached, the curves for all of
the three cell lines were sigmoidal in shape. The
E-C data for the 3 cell lines were computer
fitted to the log-linear and sigmoidal
Emax models, and the estimates for
n, Emax,
IC50, and IC90 were
obtained (Fig. 3)⇓
. Table 1⇓
lists the fitted PD parameters of the three cell lines. The
IC50s for a 4-hour exposure were 23.28
nm (MGH-U1), 33.51 nm
(MCF-7), and 126.51 nm (HT-29). The
IC50s for exposures of 24 h or longer were
similar among the three cell lines. When looking at
IC90s, MGH-U1 was the most sensitive line and
MCF-7 the most resistant. The range of concentration required to kill
one log of cells in the different lines was 11.6–113.5
nm for a 24-h exposure and only 9.9–14.3
nm for a 72-h exposure. The cytotoxicity of 9-AC
to the three cell lines increased with longer exposure time. However,
the cytotoxicity was limited in the case of shorter duration of
exposure (Fig. 2)⇓
. When HT-29 and MCF-7 cells were exposed to 9-AC for
less than 24 h, less than 1 log of cell killing occurred, even at
the highest concentrations (274 nm). Increasing
the exposure time from 4 h to 24 h decreased the
IC50 6-fold for the MGH-U1, 9-fold for the MCF-7,
and 21-fold for the HT-29 cell lines. An increase in the exposure time
from 24 to 72 h only slightly decreased the
IC50s for the three cell lines; however, the
concentration required to achieve 1 log of cell kill decreased from
11.6 to 9.9 nm, 113.5 nm to
14.3 nm, and 46.8 to 14.3
nm in MGH-U1, MCF-7, and HT-29, respectively.
This indicates that exposure time beyond a threshold is a critical
determinant of 9-AC cytotoxicity.

Values for IC50 and IC90, and n, and
Emax were determined from the computer fit of
experimental data using sigmoidal Emax model.
r2 (goodness of fit from the correlation of
observed values to the model predicted values) was ≥0.99 for
all of the exposure times in all of the cell lines.

The cytotoxicity of 9-AC to the three cell lines also increased with
higher drug concentrations. However, minimum cell killing was observed
until concentrations greater than 2.7 nm were used. When
9-AC concentration was increased from 2.7 to 13.7 nm, great
changes in cytotoxicity were observed for the MGH-U1 and HT-29 cell
lines. Additional increases above 13.7 nm did not
significantly change the cytotoxicity, even with longer exposures.

PK/PD Modeling of 9-AC.

The AUC of 9-AC for each concentration at different exposure times was
calculated using LAGRAN V1.0B software. The relationship between the
AUC of 9-AC lactone and the cytotoxicity of 9-AC was then analyzed.
Table 2⇓
lists comparisons on the basis of similar AUC values for different time
points and concentrations and the survival percentages of the three
cell lines. With a similar AUC, the HT-29 cell line survival
percentages ranged from 68 to 77%, 8 to 51%, and 3 to 27%. The same
results were observed in the other two cell lines, which indicated no
fixed relationship between AUC and survival percentage could be
modeled.

The AUC for 9-AC for different exposure points
and their cytotoxicity for three cell lines

AUC for 9-AC was calculated using LAGRAN V1.0B software. Survival
percentage of three cell lines was obtained from cytotoxicity studies.

The data from the multiple exposure time experiments for the three cell
lines were then fitted to the PD model
Cnt = k. Using
this equation, we determined the PD parameters n and
k from a plot of the isotoxic concentrations
IC50 or IC90versus time of exposure using least squares nonlinear
regression fitting. Cmin,
tmin, and (C ×
t)min were then calculated from the
n and k values. All of the parameters for the
three cell lines are summarized in Table 3⇓
. For 50 or 90% of cell killing, the cytotoxicity-drug
concentration-exposure time (E-C-t)
relationship could be fitted very well by this model with
r2
ranging from 0.90 to 0.99. To kill
90% of cells, n values ranged from 0.3 to 0.85 for all
three cell lines, which indicated the relatively greater importance of
exposure time to cytotoxicity. The
Cmin,
tmin, and (C ×
t)min values represented the critical
exposure time and drug concentrations required for a certain drug
effect and thereby provided a useful PD framework for additional
in vitro studies. To reach 90% cell killing, a
longer-than-24-h exposure was necessary for all three of the cell lines
(tmin = 50, 35, and 24 h,
respectively), and Cmin was 25, 29,
and 7 nm, respectively.

Parameters n, k, r2 were determined by computer
fitting the IC50 or IC90 data to the PD
relationship Cn × t = k.
r2 is the goodness of fit from the correlation of
observed values to the predicted values. Cmin,
tmin, and (C ×
t)min were calculated from the n and
k values as described in the “Materials and Methods”
section.

Effects of 9-AC on Cell Cycle Progression.

The cell cycle effects of 9-AC were studied as a function of
concentration and time (Table 4)⇓
. Consistent with the cytotoxicity studies, 4-h exposure to 9-AC at
both 13.7 nm and 137 nm had no demonstrable
effects on cell cycle in any of the three cell lines. However, after
24-h exposures to 13.7 nm 9-AC, the HT-29 and MCF-7 cell
lines arrested in G2-M and remained there for
72 h after drug exposures. A G2-M arrest was
transient in the MGH-U1 cell line, in which cells re-entered into the
cell cycle at 48 h and displayed DNA histograms similar to the
controls at 72 h. After 24-h exposure to 137 nm 9-AC,
all three cell lines accumulated in S phase. The MGH-U1 and MCF-7 cells
remained in S phase at 72 h, whereas the HT-29 cells progressed
through S phase and arrested in G2-M 48 h
after drug exposure and remained there at 72 h.

Each value represents the mean ± SD of three or more independent
experiments.

DISCUSSION

Because the active 9-AC lactone is unstable in aqueous solution,
the kinetics of 9-AC lactone need to be studied to accurately determine
the relationship between cytotoxicity and the AUC in vitro.
The total concentration of 9-AC was also monitored to ensure that the
concentration of 9-AC was constant through the process of cell killing.
At equilibrium, the 9-AC lactone:9-AC carboxylate ratio in media was∼
35:65, which was higher than that reported in human plasma (9:91;
Ref. 28
). Human serum albumin exhibits a 200-fold binding
preference for the carboxylate relative to the lactone. The binding
preference of the 9-AC carboxylate may accelerate the hydrolysis and
shift the equilibrium favoring the formation of carboxylate, hence
decreasing the 9-AC lactone percentage at equilibrium in human plasma.

Although there were differences in sensitivity of the 3 cell lines to
9-AC, these differences became less pronounced during longer exposure
times. The MGH-U1 cell line had the shortest doubling time and was the
most sensitive cell line among the three cell lines studied in our
experiment (doubling times were 18, 28, and 36 h for MGH-U1,
MCF-7, and HT-29, respectively). The CPTs are S-phase-specific agents
and cell lines with a high S-phase fraction should be more sensitive to
the drug (29, 30)
. Although we did see some differences in
doubling times, there were no significant differences in S-phase
fraction between the three lines. A threshold concentration of at least
2.7 nm 9-AC was required for any drug cytotoxicity, whereas
a minimum of 7–29 nm was required for 1 log of cell kill.
An increase in 9-AC concentration from 2.7 to 13.7 nm
dramatically decreased the survival percentage for all of the three
cell lines. A further increase in concentration above 13.7
nm only slightly decreased the survival fraction. These
observations are in agreement with a previously reported preclinical
study of 9-AC and suggest that maintenance of the 9-AC lactone within a
critical threshold concentration range is important for efficacy
against human cancers in vitro and in vivo(31)
. Although 9-AC cytotoxicity was related to
concentration with a threshold being defined, the duration of exposure
appeared to be more important. One log of cell killing occurred only
when the cells were exposed for 24 h or longer. Extension of the
duration of exposure beyond 24 h was more important for the more
resistant lines than for the sensitive MGH-U1, in which little further
increase in cytotoxicity occurred. These observations, which indicate
that 9-AC-induced cytotoxicity required a threshold duration
of drug exposure, is in accord with studies using other
S-phase-specific antitumor agents such as hydroxyurea
(32)
. The PD model, Cn ×
t = k, was able to fit the cytotoxicity
results very well; to kill 90% of cells, the value of n
ranged from 0.3 to 0.85. An n value of <1 suggests
exposure time is more important than concentration, and these data,
thus, indicate that exposure time is more important than concentration
to achieve significant cytotoxicity. The flow cytometric analysis of
cell cycle after drug exposure also supports the need for a minimum
duration of drug exposure to achieve cytotoxic effects. These findings
are consistent with those for other phase-dependent anticancer agents
such as the antimetabolites, for which duration of exposure is more
important than peak concentration (33, 34, 35)
. The
time-dependency of 9-AC-induced toxicity seen in this study is also
consistent with preclinical studies done of this drug. In a xenograft
model, 9-AC by s.c. injection was toxic, and the implanted human cancer
tumor regressed only partially (36)
. At identical dose
levels, when 9-AC was given s.c. by depot injection with gradual
release of the drug into the bloodstream, the treatment was without
apparent toxicity and resulted in complete regression of implanted
tumors.

Whether a schedule-specific therapeutic advantage can be obtained with
the use of 9-AC is not yet determined. 9-AC schedules tested in Phase I
studies reported to date are a 24-h infusion given weekly, a 72-h
infusion given every 2–3 weeks, a 5–7-day infusion every 3 weeks, and
a daily oral schedule (37, 38, 39, 40)
. The dose-limiting toxicity
in most studies is myelosuppression with steady-state concentrations of
9-AC lactone of ∼10, 2.6, and 0.5 nm at the maximally
tolerated dose of 1.65 mg/m2/day i.v., 0.84 mg/m2/day i.v., and 0.84
mg/m2/day p.o. in the 24-h, 72-h, and daily oral schedules
respectively. Using a higher dose regimen, a 7-day infusion schedule in
leukemic patients was able to achieve steady-state concentrations of 10
nm; however, severe, prolonged myelosuppression and
mucositis were associated with this schedule. The studies we have
performed suggest that the current strategy of using longer infusions
of 9-AC is a reasoned approach to the clinical development of this
drug. However, the use of infusions of longer than 24 h is leading
to steady-state plasma concentrations at the maximally tolerated dose
that are at, or below, the threshold for cytotoxicity in
vitro. Thus, the use of shorter (i.e., 24 h)
infusions of the drug, given repeatedly, may offer the greatest
opportunity for clinical benefit.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.